Three immobilized enzymes acting in series in layer by layerassemblies: Exploiting the trehalase-glucose oxidase-horseradishperoxidase cascade reactions for the optical determination oftrehalose

A new optical biosensor for trehalose determination has been realized immobilizing three glycoenzymeson a transparent support. Trehalase, glucose oxidase and horseradish peroxidase have been alternatedwith layers of Concanavalin A by a “layer-by-layer” (LbL) deposition. The driving force of this assembly isthe biospecific complexation between Concanavalin A and sugar residues in the glycoenzymes. As con-firmed by UV–vis spectroscopy, the LbL deposition allowed a high ordinate architecture with high loadingof enzymes. After the assembly, the functionality of immobilized enzymes was spectrophotometricallyproven, demonstrating also that they can act in series catalyzing cascade reactions.The prepared biosensor was used to optically detect trehalose, giving a LOD of 10 M and a linearresponse up to 4 mM, and it showed also good time stability. The trehalose content in a real sample(eyewash) was successfully determined by the biosensor.